Cleaning composition comprising microbial lipase SD2 and sodium dodecylbenzene sulfonate

ABSTRACT

This invention is directed to a detergent composition comprising the microbial lipase SD2 and dodecylbenzene sulfonate. In the detergent composition, the lipase SD2 is characterized by having (i) optimum pH for activity of about 8±0.5; (ii) an optimum temperature for activity of about 30° to 55° C. and (iii) a molecular weight as measured by gel permeation chromatography of about 8.8×10 4 .

This application is a continuation-in-part of co-pending U.S.application Ser. No. 07/324,062, filed on Mar. 16, 1989.

The invention herein described relates generally to a new detergentcomposition, and more particularly a composition suitable for use inlaundry and/or dishwashing applications.

By way of background, dodecylbenzene sulfonate ("DBS") is a commonlyused surfactant employed in household detergents. It is consideredlow-cost, safe and effective. Because of dodecylbenzene sulfonate'swide-spread usage in cleaning products, compatibility with and efficacyin the presence of this surfactant is an important consideration in theevaluation of new detergent additives.

Recently, lipases have become of interest as laundry detergentadditives. By way of illustration, Novo Industri A/S has recentlyintroduced into the marketplace a lipase referred to as LIPOLASE.However, the present inventors have found that LIPOLASE is not aseffective as might be desired in performing its function of breakingdown lipids into fatty acids, particularly in the presence of DBS whenformulated into dodecylbenzene sulfonate-containing launderingformulations.

In view of the above, new lipases exhibiting enhanced efficacy in thepresence of dodecylbenzene sulfonate would be highly desired by thedetergent manufacturing community.

In one aspect, the present invention relates to a detergent compositioncomprising the microbial lipase SD2 and sodium dodecylbenzene sulfonate.In the detergent, the lipase is characterized by having (i) an optimumpH for activity of about 8±0.5; (ii) an optimum temperature for activityof about 30° to 55° C. and (iii) a molecular weight as measured by gelpermeation chromatography of about 8.8 ×10⁴. This and other aspects willbecome apparent from a reading of the following detailed specification.

The present inventors have isolated a biologically pure culture of apreviously undescribed strain of Pseudomas alcaligenes, strain SD2, asdisclosed and claimed in co-pending, commonly-assigned U.S. applicationSer. No. 324,062, incorporated herein by reference in its entirety. Theorganism is a natural isolate and has been deposited with the AmericanType Culture Collection (ATCC), having been assigned the accessionnumber ATCC 53877. This novel strain SD2 was found to produce a novellipase.

The microorganism, P. alcaligenes, strain SD2, was isolated from ashower drain by direct isolation on a Tryptone-Soytone-Olive oilisolation medium. The isolation medium employed is more fully describedin Table I below.

                  TABLE I                                                         ______________________________________                                        Isolation Medium                                                                                Percent by Weight                                           ______________________________________                                        Ammonium sulfate    0.5                                                       Potassium phosphate, dibasic                                                                      0.05                                                      Magnesium sulfate, heptahydrate                                                                   0.025                                                     Tryptone (Difco)    1.7                                                       Soytone (Difco)     0.3                                                       Olive oil           1.0                                                       Rhodamine B         0.001                                                     Agar                1.5                                                       ______________________________________                                    

The Rhodamine B dye in the isolation medium causes lipase-producingbacterial colonies to fluoresce an orange color when irradiated withlong wavelength ultraviolet light (Kouker, G. and K. -E. Jaeger, 1987,App. Environ. Microbiol, 53: 211-3). This fluorescence permits the easyidentification of lipase-producers. Colonies so identified were purifiedby restreaking onto similar media. Stock cultures were maintained onDifco TSA slants.

The bacterial isolate was identified using standard taxonomic proceduresfrom Bergey's Manual of Systematic Bacteriology (Williams & Wilkins,Baltimore, 1984). The results of applicable physiologicalcharacterization tests of P. alcaligenes strain SD2 are presented inTable II and compared with characteristics of P. alcaligenes and P.pseudoalicaligenes published in Bergey's Manual.

                  TABLE II                                                        ______________________________________                                        Substrate Utilization of P. alcaligenes                                       Strain SD2, P. alcaligenes, and P. pseudoalicaligenes                                       Strain*                                                                  SD2    P. alcaligenes                                                                           P. pseudoalicaligenes                              ______________________________________                                        Fructose   -        -          +                                              L-aspartate                                                                              +        -          -                                              L-glutamate                                                                              -        +          +                                              D-gluconate                                                                              -        -          d                                              L-Histidine                                                                              -        d          d                                              Ethanolamine                                                                             -        -          +                                              n-Butanol  -        d          +                                              Isobutanol +        d          -                                              Citrate    -        d          d                                              Betaine    -        -          +                                              Glycerol   -        -          d                                              Sorbitol   -        -          d                                              Itaconate  -        -          d                                              ______________________________________                                         Abbreviation:                                                                 d (11-80 percent of strains positive);                                        + (strain was able to utilize the indicated chemical for growth);             - (strain did not utilize the chemical for growth).                           *Data for P. alcaligenes and P. pseudoalicaligenes are from Bergey's          Manual of Systematic Bacteriology (Williams & Wilkins [Baltimore, 1984]).     Compounds utilized by all strains include: DLlactate, succinate, fumarate     acetate, Larginine, caprate, and Lmalate.                                     Compounds not utilized by any strain include: Dglucose, Larabinose,           Dmannose, Dmannitol, Lrhamnose, D(+)galactose, D(-)ribose, m. inositol,       Lthreonine, mtartrate, adipate, phenylacetate, nicotinate, sebacate,          suberate, benzoate, and pimelate.                                        

This table illustrates nutritional capabilities of the indicated strainsand further illustrates their differences.

Several lipase-producing strains of P. pseudoalicaligenes are disclosedin International Publication No. WO 87/00859 published under the PatentCooperation Treaty. Table III presents certain morphological andphysiological characteristics of P. alcaligenes strain SD2, as comparedto the characteristics of four strains of P. pseudoalicaligenesdisclosed in International Publication No. WO 87/00859. Differencesbetween the SD2 strain of the present invention and the other strainsare readily apparent. For example, SD2 utilized L-aspartate, while thetwo other Pseudomonas species did not, as noted noted in Table II.

                  TABLE III                                                       ______________________________________                                        Characteristics of P. alcaligenes Strain SD2 and                              Selected Lipase-Producing Strains of P.                                       pseudoalicaligenes.                                                           (The CBS Strain Accession Numbers Correspond to Those                         Referenced in International Publication No. WO 87/00859)                                   Comparison Strains                                                         Strain                                                                        of                                                                            Invention                                                                              CBS       CBS   CBS   CBS                                  Characteristic                                                                          SD2      467.85    468.85                                                                              471.85                                                                              473.85                               ______________________________________                                        Cell shape                                                                              rod      rod       rod   rod   rod                                  Motility  +        +         +     +     +                                    Spores    -        -         -     -     -                                    Gram Strain                                                                             -        -         -     -     -                                    Oxidase   +        +         +     +     +                                    Anaerobic -        -         -     -     -                                    glucose                                                                       Aerobic   -        -         -     -     -                                    glucose                                                                       Aerobic   -        -         -     -     -                                    maltose                                                                       Aerobic   -        -         -     -     -                                    sucrose                                                                       Aerobic   -        -         -     -     +                                    D-xylose                                                                      Arginine  +        +         +     -     +                                    dihydralase                                                                   Gelatin   -        -         -     -     -                                    hydrolysis                                                                    Starch    -        -         -     -     -                                    hydrolysis                                                                    NO.sub.3.sup.- →NO.sub.2.sup.-                                                   +        +         +     +     +                                    NO.sub.2.sup.- →N.sub.2                                                          +        -         -     -     -                                    Citrate   -        +         +     +     +                                    Utilization                                                                   Catalase  +        +         +     +     +                                    Growth at +        +         +     +     +                                    41° C.                                                                 ______________________________________                                    

Strain SD2 of the present invention can be grown in various types ofculture media under conditions suitable for growth of pseudomonads.Typically, such media contain assimilable sources of carbon, nitrogen,and various inorganic mineral nutrients. By way of illustration, P.alcaligenes strain SD2 was grown in L-Aspartate Medium having theformulation as shown in Table IV.

                  TABLE IV                                                        ______________________________________                                        Culture Medium                                                                Ingredient            Percent by Weight                                       ______________________________________                                        Ammonium sulfate      0.5                                                     Potassium phosphate, dibasic                                                                        0.05                                                    Magnesium sulfate, heptahydrate                                                                     0.025                                                   Tris(hydroxymethyl)aminomethane                                                                     1.21                                                    L-Aspartic acid       2.0                                                     Brij ® 58         1.0      mM                                             FeCl.sub.3            1.0      μM                                          ______________________________________                                    

The medium is adjusted to pH 7.5-8.0 with potassium hydroxide prior tosterilization. The advantage of this medium over the Tryptone mediumreferred to in U.S. application Serial No. 324,062 is that a whiteproduct is obtained, free of colored high molecular weight metabolitestypically found in Tryptone medium.

The lipase of the invention is found in culture media, preferably liquidmedia, containing P. alcaligenes strain SD2. Quantities of this enzymecan be obtained by culturing P. alcaligenes strain SD2 in liquid cultureand under culture conditions suitable for growth of organisms of thistype. For example, an actively growing broth culture of P. alcaligenesstrain SD2 is suitably used as an inoculum and introduced intoErlenmeyer flasks containing L-Aspartate medium (C. F. Table IV). Inaddition, the inclusion of the non-ionic surfactant BRIJ® 58[polyoxyethylene (20) cetyl ether] in liquid growth medium containing P.alcaligenes strain SD2 at a 1-10 mM concentration, preferably 1 mM,increased the yield of the lipase by a factor of two-fold or more incontrast to control cultures without this surfactant. Cultures areincubated with shaking for about 24 hours at a temperature of about 30°C. Following this culture growth period, the bacterial cells are removedby centrifugation or filtration or other suitable techniques. The lipasewhich is found in the resultant clarified culture liquor is thengenerally concentrated prior to use. Several methods may be used toconcentrate this enzyme, including ultrafiltration as discussed inExample 1.

It is desirable that lipases intended for commercial utilization bestable in the presence of various surfactants commonly found in cleaningproduct formulations. Advantageously, the lipase of P. alcaligenesstrain SD2 was found to be functional in the presence of commercialsurfactants such as dodecylbenzene sulfonate and fatty alcoholethoxysulfates. In a laundry detergent composition the lipase strain SD2is employed in an amount of between about one million and about 100million, preferably between about 5 and about 10 million lipase unitsper kilogram of DBS in the detergent. Upon dilution of the detergentcomposition with water to form a wash solution, the lipase SD2 isgenerally present in an amount of between about one and about 500,preferably between about 3 and about 5 lipase units per milliliter oflaundry wash solution. The term "lipase unit" is defined in Table V,footnote (1).

Regarding the stability of the lipase produced by P. alcaligenes strainSD2, this enzyme loses activity during storage at a rate that isdirectly proportional to temperature. For example, during acceleratedaging tests conducted at a temperature of 37° C. and a pH of 7.0, thelipase useful in this invention demonstrated a half-life of about 5 daysin the absence of surfactants. The addition of calcium, in the form ofCaCl₂, stabilized the SD2 lipase and increased its half-life to over 45days at suitable CaCl₂ concentrations. The concentration of CaCl₂required to enhance such enzyme longevity is related to the particularlipase formulation. For example, in simple buffered enzyme solutionslacking surfactants, where the buffer is, for example, 50 mM BES [N,N-bis (2-hydroxyethyl)-2-amino-ethanesulfonic acid] at pH 7.0, theaddition of 5 mM CaCl₂, preferably 10 mM, is sufficient. The optimumconcentration of CaCl₂ in the presence of preferred surfactants is about25 mM or more. In formulations of the lipase of P. alcaligenes strainSD2, various surfactants can be used in view of this lipase's stabilityin the presence of surfactants. Examples of preferred surfactantsinclude the non-ionic surfactant BRIJ® 35 [polyoxyethylene (23) laurylether] and the anionic surfactant SANDOPAN® DTC gel (sodiumtrideceth-7-carboxylate). Preferred non-ionic surfactants are thosehaving a hydrophobic end containing 12-16 carbon units, and apolyoxyethylene chain size of about 20-23 ethylene oxide units. Ingeneral, anionic surfactants of the carboxylated type are preferred andare most compatible with the novel lipase of P. alcaligenes strain SD2.

While the invention has been described above with reference to specificembodiments thereof, it is apparent that many changes, modifications andvariations can be made without departing from the inventive conceptdisclosed herein. Accordingly, it is intended to embrace all suchchanges, modifications and variations that fall within the spirit andbroad scope of the appended claims. All patent applications, patents andother publications cited herein are incorporated by reference in theirentirety.

EXAMPLE 1 (A) Preparation of Lipase From Pseudomonas alcaligenes StrainSD2

The microorganism of the invention, P. alcaligenes SD2, was convenientlygrown in the culture medium previously presented in Table IV.

A 50 mL starter culture of P. alcaligenes SD2 in a 250 mL Erlenmeyerflask was grown for about 16 hours at a temperature of 30° C. at 175 rpmon a gyratory shaker. This starter culture was then used to inoculate 8liters of culture medium divided among 4 and 6 L fluted Erlenmeyerflasks such that no individual flask contained more than 25 percentflask capacity as liquid. The culture flasks thus prepared wereincubated for 24 hours at a temperature of 30° C. with gyratory shakingat 150 rpm.

Following the culture period, the lipase of the invention was harvestedand concentrated by first removing the bacterial cells from the 8 litersof liquid culture by tangential flow filtration using Pharmacia 10⁶(NMWC) Omega membrane cassettes. The resultant cell-free filtrate wasthen concentrated by tangential flow ultrafiltration using Pharmacia30,000 (NMWC) Omega membrane cassettes. Thereafter, the concentrate wasdiafiltered at 3° C. with about 10 volumes of 50 mM BES, pH 7.0,supplemented with 10 mM CaCl₂ in order to eliminate all low molecularweight contaminants (those with molecular weights less than or equal to30,000), and to change the lipase solvent to one with buffer andstabilizing CaCl₂. The yields of enzyme from three separate batchcultures are presented in Table V.

                  TABLE V                                                         ______________________________________                                        Yields of Lipase Produced by Cultures                                         of P. alcaligenes Strain SD2                                                  Batch No.      Units/mL.sup.(1)                                                                        Total Units                                          ______________________________________                                        20             39.15     10,571                                               21             34.69     7,840                                                22             37.41     6,172                                                ______________________________________                                         .sup.(1) One unit is the amount of lipase which produces one                  microequivalent of fatty acid from olive oil per minute at 37° C.      and at pH 10.                                                            

(B) Production of the Lipase P. alcaligenes Strain SD2 and MolecularWeight Measurement

Quantities of the lipase of P. alcaligenes strain SD2 were obtained byculturing of the organism in the medium of Table IV, removing thebacterial cells by filtration, concentrating the enzyme byultrafiltration as already described. Lipolytic activity was assayedusing the following standard composition: (i) 2.5 mL substrate [10percent (w/v) olive oil emulsified in 10 percent (w/v) gum arabic]; (ii)2.0 mL buffer [1.0M CHES (2[N-cyclohexylamino]-ethane sulfonic acid), pH10.0]; (iii) enzyme; and (iv) distilled water added for a final volumeof 6.0 mL. Enzymatic assays were conducted at a temperature of 37° C.The fatty acids formed during the hydrolytic enzymatic reaction wereextracted with an organic solvent and titrated following the proceduredescribed in U.S. Pat. No. 4,283,494.

A quantity of the lipase of the invention was used to determine itsmolecular weight. The molecular weight of the lipase of P. alcaligeneswas found to be about 88,000 using gel filtration chromatography andcomparing the retention time of the lipase with molecular weightcalibration standards.

(C) Laundering Effectiveness of P. alcaligenes SD2 And DodecylbenzeneSulfonate

Laundering effectiveness of SD2 lipase was evaluated in a standardizedprocedure adapted from one disclosed in European Patent Application0214761 #86306091.9 (6/8/86), incorporated herein by reference in itsentirety. The ingredients in the laundering solution and the procedurefollowed in the laundering protocol are described below.

Laundering Solution

(a) 0.2M Tris HCl, pH 8.5

(b) Lipase, 5 units/mL (determined at pH 8.5, 30° C.)

(c) Surfactant, 0.05% (w/v) sodium dodecylbenzene sulfonate based uponthe volume of the laundering solution

(d) Water added to provide a final solution volume of 10.0 mL

Laundering Protocol

The tests were conducted at 40° C. for the times indicated. Cleaningefficacy was determined by measuring the amount of fatty acids producedas a result of triglyceride hydrolysis, expressed as a percentage ofavailable fatty acids added as triglyceride to the fabric. Triglyceridestained fabric is prepared by adding 0.5 mL of a 3.0% (v/v) lard oil inchloroform solution to each side of a 2"×3" swatch of No. 400MMercerized white cotton fabric (Testfabrics, Inc., Middlesex, N.J.).

The stained cloths are cut into 8 similar size pieces then placed in25×150 mm test tubes containing the laundering solution. For the15-minute surfactant exposure, the laundering proceeds for 60 minutes inthe presence of the enzyme before surfactant is added and laundering iscontinued for an additional 15 minutes. The surfactant is presentthroughout the 75 minutes of laundering in the 75-minute test. The tubesare agitated on a vortex mixer for 10 seconds every 5 minutes for theduration of the test. After completion of the laundering period, samplesof the laundering solution are taken for fatty acid analysis which isperformed using the NEFA-C test kit produced by Wako Pure ChemicalIndustries, Ltd. (Osaka, Japan). Previous tests have shown this test kitprocedure is not interfered with by the ingredients of the launderingsolution at the concentrations used.

The results in terms of cleaning efficacy for two trials, using twodifferent batches of SD2 lipase were compared against the resultsobtained using similar amounts of NOVO Lipolase™, a commercial lipaseintended for use as a laundering detergent additive. With SD2 lipase,cleaning increased with time of surfactant exposure. In contrast,cleaning decreased with exposure to the surfactant in the case of NOVOLipolase. Percent cleaning ranged from 13% to 23% in the "15-minutetest" using SD2 lipase to 22% to 35% cleaning in the "75-minute test."By contrast, NOVO Lipolase exhibited no better than 8% cleaning in the"15-minute test" and 2% to 3% in the "75-minute test."

Based upon the above results, it is clear that SD2 lipase is effectivein removing triglyceride stains from cotton fabric in the presence ofthe commonly used dodecylbenzene sulfonate surfactant at a concentrationof the latter that is commonly encountered in laundering. SD2 lipase isclearly superior to NOVO Lipolase in these comparisons and, on the basisof these results, would be expected to outperform NOVO Lipolase informulations containing this anionic surfactant.

What is claimed is:
 1. A detergent composition comprising the microbiallipase SD2 and dodecylbenzene sulfonate, wherein said lipase is presentin an amount of between about one million and about 100 million lipaseunits per kilogram of dodecylbenzene sulfonate in the detergentcomposition, and wherein said dodecylbenzene sulfonate is present in anamount of between about 0.01 and about 20 weight percent based upon thetotal weight of said detergent composition.
 2. The detergent compositionof claim 1 wherein said lipase SD2 is characterized by having (i) anoptimum pH for activity of about 8±0.5; (ii) an optimum temperature foractivity of about 30° to 55° C. and (iii) a molecular weight as measuredby gel permeation chromatography of about 8.8×10⁴.
 3. The detergentcomposition of claim 1 wherein said lipase is present in an amount ofbetween about 5 million and about 10 million lipase units per kilogramof dodecylbenzene sulfonate in the detergent composition.
 4. In animproved detergent composition comprising dodecylbenzene sulfonate, theimprovement comprising said detergent additionally containing themicrobial lipase SD2 in a cleaning effective amount of between about onemillion and about 100 million lipase units per kilogram ofdodecylbenzene sulfonate in the detergent composition, saiddodecylbenzene sulfonate being present in an amount of between about0.01 and about 20 weight percent based upon the total weight of saiddetergent compositon.
 5. A laundry wash solution comprising water,dodecylbenzene sulfonate and the microbial lipase SD2, saiddodecylbenzene sulfonate being present in a cleaning effective amount ofbetween about 0.01 and about 20 weight percent based upon the totalweight of said wash solution and said lipase being present in said washsolution in an amount of between about 1 and about 500 lipase units permilliliter of wash solution.
 6. The laundry wash solution of claim 5wherein said lipase is present in said wash solution in an amount ofbetween about 3 and about 5 lipase units per milliliter of washsolution.